AGPase catalyzes the first committed step in starch (plants) and glycogen (bacteria) synthesis. It involves the conversion of glucose-1-P (G-1-P) and ATP to ADP-glucose and pyrophosphate (PPi). AGPase is a heterotetramer in plants consisting of two identical small and two identical large subunits. The large and the small subunits are encoded by shrunken-2 (Sh2) and brittle-2 (Bt2) respectively in maize endosperm. AGPase is allosterically regulated by small effector molecules that are indicative of the energy status of the cell. AGPase is activated by 3-PGA, the first carbon assimilatory product, and inhibited/deactivated by inorganic phosphate (Pi) in cyanobacteria, green algae and angiosperms.
The importance of maize endosperm AGPase in starch synthesis has been shown by the kernel phenotype of mutants in either subunit of the enzyme. Indeed, such mutants result in shrunken kernels and a large reduction in endosperm starch content (Tsai and Nelson, 1966; Hannah and Nelson, 1976). There is also evidence that AGPase catalyses a rate-limiting step in starch synthesis (Stark et al. 1992; Giroux et al. 1996; Greene et al 1998b; Sakulsingharoja et al. 2004; Obana et al. 2006; Wang et al. 2007).
Greene and Hannah (1998a) isolated a mutant form of maize AGPase with a single amino acid change in the large subunit termed HS33. They showed that the altered enzyme was more heat-stable and that stability was due to stronger subunit-subunit interactions. When wheat and rice were transformed with a Sh2 variant that contains the HS33 change along with a change that affects the allosteric properties of AGPase (Rev6)(Giroux et al., 1996), yield was increased by 38% and 23% respectively (Smidansky et al., 2002; 2003). Remarkably, the increase was due to an increase in seed number rather than individual seed weight.
Transformation of maize with the Sh2 variant containing the Rev6 and HS33 changes also gives rise to enhanced seed number. Seed yield/ear can be increased up to 68% in maize. Enhanced seed number cannot be explained by Rev6 since, when expressed alone in maize, it increases only seed weight (Hannah, unpublished). The above studies show the importance of AGPase heat stability in cereal yield.
Cross et al. (2004) generated a mosaic small subunit (MP) consisting of the first 200 amino acids of BT2 and the last 275 amino acids of the potato tuber small subunit. MP in a complex with SH2 had several features that could lead to agronomic gain (Cross et al., 2004; Boehlein et al., 2005). Some of those features were increased activity in the absence of the activator 3-PGA, increased affinity for 3-PGA and elevated heat stability compared to wildtype maize endosperm AGPase (BT2/SH2). Preliminary data show that maize plants with transgenic MP containing AGPase variant expressed in maize endosperm provides for a starch yield increase (Hannah, unpublished data).